Method for reducing serum lipoprotein (A) concentration

ABSTRACT

A method of reducing serum Lp(a) concentration in a subject is accomplished by administration of liposomes. A suspension of small, unilamellar vesicles composed primarily of phosphatidylcholine phospholipids is administered parenterally to a subject having or at risk for developing a disease condition associated with an elevated serum Lp(a) concentration. The liposomes are infused over a period of time such that a significant drop in serum Lp(a) concentration is observed.

This application is a continuation of application Ser. No. 08/522,745,filed Aug. 31, 1995.

FIELD OF THE INVENTION

The present invention relates to a therapeutic lipoprotein compositioncontaining lipoprotein particles formed from small unilamellar vesiclesand A and C apoproteins and methods for its formation and use.

REFERENCES

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Harrison, T. R., Ed., HARRISON'S PRINCIPLES OF INTERNAL MEDICINE,TWELFTH ED. pp. 839, 1814-1825, McGraw Hill, Inc. (1991).

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Rifici, V. A., et al., Biochim. Biophys. Acta 834:205-214 (1985).

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BACKGROUND OF THE INVENTION

Lipoproteins are high molecular weight particles that are primarilyresponsible for lipid transport, namely of triglycerides and cholesterolin the form of cholesteryl esters, through the plasma. Five majorclasses of naturally-occurring lipoproteins are known to circulate inplasma, each differing in lipid composition, apoprotein composition,density, size, and electrophoretic mobility.

Each lipoprotein particle is composed of a non-polar core region, asurrounding phospholipid surface coating containing small amounts ofcholesterol, and exposed at the surface, apoproteins responsible forbinding to receptors on cell membranes and directing the lipoproteincarrier to its intended site of metabolism.

At least ten different apoprotein molecules have been identified, andeach class of lipoprotein particle contains a specific apoprotein (alsoreferred to as apolipoproteins) or combination of apoproteins embeddedin its surface (Harrison). These apoproteins are encoded by geneslocalized to sites on chromosomes 1, 2, 6, 11, and 19, and mutationsthereof are thought to play a role in atherogenesis.

The major classes of lipoproteins found in human plasma includechylomicrons and chylomicron remnant particles, VLDL (very low densitylipoprotein), IDL (intermediate density lipoprotein), LDL (low densitylipoprotein), and HDL (high density lipoprotein).

Chylomicrons contain a hydrophobic core primarily composed of dietarytriglycerides and contain several apoproteins including AI, AII, B48,CI, CII, CIII, and E. VLDL contains a core of endogenous triglyceridessynthesized in the liver, in addition to apoproteins B48, CI, CII, CIII,and E. IDL particles are composed of lipids including cholesteryl estersand triglycerides and contain apoproteins B100, CIII, and E. A fourthclass of lipoprotein, LDL, possesses a core composed almost entirely ofcholesteryl esters and has a surface coat containing only apo B100.About three-fourths of the total cholesterol in normal human plasma iscontained within LDL particles. A fifth class of lipoprotein, HDL, alsocontains cholesteryl esters and possesses a surface coating whichincludes AI and AII apoproteins. A detailed description of the majorclasses of human lipoproteins and their function in lipid transport isprovided in Harrison (Harrison, 1991).

In addition to the major classes of lipoproteins, a lipoprotein-likeparticle, Lp(a), has been identified and shown to bear a strongresemblance to both lipoprotein and plasminogen. Its protein componentsinclude apo B100 linked to apo (a) via a disulfide bridge. Although arelationship has not been clearly established, the structuralresemblance of the lipoprotein-like Lp(a) particle to plasminogen isthought to provide a link between lipids, the clotting system, andatherogenesis.

HDL has been shown to be associated with a protective effect againstatherosclerosis in humans (Miller, 1975; Blankenhorn, 1987). It has beenhypothesized that HDL exerts its protective effect by the reversetransport of excess cholesterol from peripheral tissues to the liver(Bailey, 1965; Glomset, 1968). However, in regard to its role inanti-atherogenesis, the mechanisms of HDL uptake of cholesterol anddelivery to the liver remain an issue of debate (Bisgaier, 1988).

In studies in rats, the vast majority of IDL is shown to be rapidlycleared by the liver. The mechanism of IDL uptake is not clearlyunderstood, although it appears to involve a receptor-mediated process(Noel, 1983).

Numerous studies have been undertaken to elucidate the specificmechanisms of lipid uptake by lipoproteins, the role of lipoproteins inatherogenesis, additional physiological roles of lipoproteins, theidentification of lipoprotein receptors responsible for cell surfacebinding, and the like. The findings resulting from such studies areoften inconclusive due to the complex nature of lipoproteins and theaccompanying complexity of the human lipid transport system, and areoften derived from in-vitro studies or are based on animal models.

SUMMARY OF THE INVENTION

The lipoprotein composition contains lipoprotein particles, according tothe invention, composed of small unilammellar vesicles ofphosphatidylcholine phospholipids, and associated with thephospholipids, are apoproteins from apoprotein classes A and C. Thephosphatidylcholine phospholipids have phase transition temperaturesbetween about -10 and 37° C. and are preferably egg phosphatidylcholinephospholipids. The particles form a therapeutic composition foradministration to a subject by intravenous administration.

The particles and composition are formed, in one embodiment, by mixingsmall unilamellar vesicles of phosphatidylcholine phospholipids with amixture of apoproteins consisting essentially of apoproteins fromclasses A and C, typically apo A-1 and C class lipoproteins. Thecomponents are mixed under conditions effective to form lipoproteinparticles in which the apoproteins are vesicle-associated. Typically,the vesicles contained in the composition have sizes between 0.03 and0.08 microns.

In one general embodiment, the mixing is carried out ex vivo, e.g., bymixing SUV's with a mixture of recombinant A and C apoproteins. Inanother general embodiment, the mixing is carried out in vivo byadministering the SUV's to a human subject, allowing the liposomes tocirculate for a period of at least about 2 hours, and then optionally,isolating a serum sample from the subject, and isolating from the serumsample. The lipoprotein particles have a density of between about 1.0006and 1.019 g/ml.

The particle composition is used, in accordance with another aspect ofthe invention, for treating a disease state which is responsive tointravenous administration of small unilammelar phosphatidylcholinevesicles, in the dosage range preferably between about 1-50 mg vesiclelipid/kg subject body weight. The treatment method includesadministering to a subject in need of such treatment, a therapeuticallyeffective dose of a lipoprotein composition, and repeating theadministering, if necessary, until a measurable improvement in thedisease state is observed.

In one embodiment the method is used in treating acute renal failure ina subject, as evidenced by a ratio of urine-to-plasma creatinine lessthan about 20, where the composition is administered until a significantincrease in urine-to-serum creatinine ratio is achieved.

In another aspect, the method is used in treating hypertension in asubject having elevated diastolic blood pressure, where the compositionis administered until a significant reduction in blood pressure, e.g., a20% reduction in diastolic blood pressure, is achieved.

In a related aspect, the invention includes a method of treating adisease state which is associated with elevated levels of Lp(a), byadministering to a subject having an elevated serum Lp(a) level, atherapeutically effective dose of the lipoprotein composition, withrepeated administration, if necessary, until a measurable reduction inserum Lp(a) level is observed.

These and other objects and features of the invention will become morefully apparent when the following detailed description is read inconjunction with the accompanying figures and examples.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Unless indicated otherwise, the terms below have the following meanings:

"Empty" liposomes refers to liposomes that do not contain entrapped orencapsulated drug.

"Recombinant apoprotein" refers to an apoprotein which is prepared bygenetic engineering techniques commonly known in the art. Recombinantapoproteins are produced typically by cloning the gene of interest intoa vector for introduction into any of a number of expression systemssuch as E. coli, yeast, cultured mammalian cells, and the like.

Small unilammelar vesicles (SUVs) refer to small single-bilayerliposomes having sizes between about 0.02 to 0.12 microns, preferably0.02 to 0.08 microns.

"Associated apoproteins" in the context of the present invention refersto apoproteins which are stably bound to the SUV's, as evidenced by theability of SUV's and associated apoproteins to co-migrate in densitycentrifugation.

A "significant improvement" in a disease state is a measurable degree ofimprovement, as indicated by either a clinical or biochemical indicator,in the disease state. Typically, a significant improvement in a diseasestate is one which results in an improvement of a parameter with a knowncorrelation to the disease state of at least five percent. For example,with respect to conditions associated with elevated serum LP(a) levels:

The term elevated Lp(a) concentration refers to a serum Lp(a)concentration above 25 mg/dl. The term chronic elevated Lp(a)concentration, as used herein, refers to a concentration of Lp(a) thatis on average elevated above normal average serum Lp(a) concentrationswhen measured at various times over the course of a week. A normalaverage serum Lp(a) concentration is generally below 25 mg/dl.

The term significant reduction in Lp(a) concentration, as used herein,refers to a reduction of at least 20%, preferably more than 40%, withrespect to the pretreatment Lp(a) concentration in a subject.

In the case of elevated blood pressure, a significant reduction in bloodpressure is a reduction of at least about 10% in diastolic pressure.

II. Preparation of Lipoprotein Composition

The present invention involves, in one aspect, a therapeutic lipoproteincomposition for use in intravenous administration to a subject. Thelipoprotein composition contains lipoprotein particles of the inventioncomposed of small unilammelar vesicles (SUVs) having associated A and Capoproteins. Preparation of the lipoprotein particles of the presentinvention is described in the Examples and in the sections which follow.

A. Preparation of Liposomes: Composition

The lipoprotein particles of the present invention are composed of smallunilammelar vesicles. In one preferred embodiment, described and used inthe examples below, the liposomes are composed predominantly (more than50 mole percent, preferably more than 80-90 mole percent) ofphosphatidylcholine (PC) having a phase transition temperature less thanabout 37° C., preferably between about -10 to 24° C., e.g., about 5° C.or less.

The lipoprotein composition used in the method of the present inventionis composed primarily of PC phospholipids. PC phospholipids includethose phospholipids having a choline moiety and where the fatty acidchain portion of the phospholipid may vary in length and degree ofunsaturation.

One preferred vesicle composition includes egg PC, which has atransition temperature of -5° C., and contains predominantly1-palmitoyl, 2-oleyl PC and 1-palmitoyl, 2-linoleyl PC. Alternately,phosphatidylcholine may be isolated from rat liver (Newman, 1961),followed by purification on alumina (Shinitzky, 1974).

The liposomes may be composed entirely of egg PC, or may contain otherlipid components which (i) are not immunogenic, (ii) do not contribute asignificant portion, i.e., more than 25-50 mole percent, of lipids withhigh phase transition temperature. Additional components may includenegatively charged lipids, such as phosphatidylglycerol (PG) orphosphatidylserine (PS). Of course, the mole percentage of these lipidsshould be relatively low with respect to PC. The liposomes may alsoinclude cholesterol or other sterols, in an amount preferably less thanabout 40 mole percent.

Lipid protective agents, such as α-tocopherol, α-tocopherol acetate, orα-tocopherol succinate, may also be included in the lipids forming theliposomes, to protect the lipid components against free radical damage(Levida). Typically such agents are included at a mole percentagebetween about 0.5% and 2%. It is advantageous to add α-tocopherol to theliposomes to maintain a balance between vitamin E and polyunsaturatedlipids in the liposomes.

B. Preparation of Unsized Liposomes

A variety of methods for producing liposomes are available, and thesehave been extensively reviewed (Szoka 1980). In general these methodsproduce liposomes with heterogeneous sizes from about 0.02 to 10 micronsor greater. As will be discussed below, liposomes which are relativelysmall and well-defined in size are preferred for use in the presentinvention, hence a second processing step for reducing the size and sizeheterogeneity of liposomal suspensions will usually be required.

In one preferred method for forming the initial liposome suspension asdescribed in Example 1, the vesicle-forming lipids are taken up in asuitable organic solvent system, preferably in a siliconized glassvessel, and dried in vacuo or under an inert gas to form a lipid film.An aqueous suspension medium, such as a sterile saline solution, isadded to the film, and the vessel is agitated (e.g., on a shaker orusing a sonicator) until the lipids have hydrated to completion,typically within about 1-2 hours. The amount of aqueous medium added issufficient to produce a final liposome suspension containing preferablybetween about 10 and 30 g lipid per 100 ml media.

During the hydration stage, the lipids hydrate to form multilamellarvesicles (MLVs) with sizes ranging between about 0.5 microns to about 10microns or larger. In general, the size distribution of MLVs can beshifted toward slightly smaller sizes by hydrating the lipids under morevigorous agitation conditions.

Example 1 describes the preparation of egg PC MLVs, followed bytreatment of the MLVs with ultrasonic irradiation to reduce the liposomesizes to produce the desired SUVs. Sizing methods for producing SUVsfrom larger multilamellar vesicles are described in further detailbelow.

The aqueous medium used in forming the liposomes may containwater-soluble agent(s) which enhance the stability of the liposomes uponstorage. A preferred stabilizing agent is an iron-specifictrihydroxamine chelating agent, such as desferrioxamine. The use of thiscompound in reducing lipid peroxidation and free radical damage indrug-containing liposomes has been reported in U.S. Pat. No. 4,797,285.Briefly, it was shown that the combination of a lipophilic free-radicalquencher, such as α-tocopherol, and the water-soluble chelator gavesubstantially better protection against lipid peroxidation damage thandid either of the protective agents alone. The chelator is included inthe aqueous medium in molar excess of the amount of free iron in themedium. Typically, a chelator concentration of between about 10-200micromolar is sufficient for reducing lipid peroxidation and freeradical damage.

C. Sizing Liposomes: SUV Preparation

The suspension of liposomes prepared as described above is preferablyfurther treated to produce liposomes having a desired size and sizehomogeneity.

The liposome suspension is generally sized to achieve a selective sizedistribution of vesicles in a size range less than about 1 micron andpreferably less than about 0.8 microns. Liposomes in this size range canbe readily sterilized by filtration through a depth filter. Smallervesicles also show less tendency to aggregate on storage, thus reducingthe potential for serious vascular blockage problems upon intravenousadministration of the final lipoprotein composition of the presentinvention. Finally, liposomes which have been sized down to thesubmicron range possess more uniform biodistribution and drug clearancecharacteristics.

Preferred liposomes are small unilamellar vesicles (SUVs), i.e.,single-bilayer liposomes having sizes between about 0.02 to 0.08microns. SUVs have been shown to possess relatively long bloodcirculation halflives, when administered intravenously, as described inco-owned U.S. patent application Ser. No. 08/257,899, filed on Jun. 10,1994. Briefly, as described therein, plots of liposome retention in thebloodstream, measured up to 1,000 minutes after IV injection, revealedthat significant quantities of liposomes remained in the bloodstreameven at 1,000 minutes.

Several techniques are available for reducing the sizes and sizeheterogeneity of liposomes, in a manner suitable for preparing the smallunilammelar vesicles of the present invention. Ultrasonic irradiation ofa liposome suspension either by bath or probe sonication produces aprogressive size reduction down to SUVs. A sonicating procedure used toproduce SUVs is described in Example 1.

Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, MLVs are recirculated through a standard emulsion homogenizeruntil selected liposome sizes, typically less than 0.1 microns, areobserved.

Extrusion of liposomes through a small-pore polycarbonate membrane is aneffective method of reducing liposome size down to a relativelywell-defined size distribution. An average range is between about 0.03and 1 micron, depending on the pore size of the membrane, such asdescribed in Example 2. Typically, the suspension is cycled through themembrane several times until the desired liposome size distribution isachieved. The liposomes may be extruded through successively smallerpore membranes, to achieve a gradual reduction in liposome size.

Liposome particle sizes can be determined by a number of techniquesincluding electron microscopy, comparative chromatography (Bisgaier,1989) and quasi-elastic light scattering.

The size-processed liposome suspension may be readily sterilized bypassage through a sterilizing membrane having a particle discriminationsize of about 0.2 microns, such as a conventional 0.22 micron depthmembrane filter. If desired, the liposome suspension can be lyophilizedfor storage and reconstituted shortly before use.

D. Apoprotein Components

The lipoprotein composition of the present invention containslipoprotein particles of the invention which are composed of SUVs asdescribed above, and associated with the SUVs, are molecules of serumproteins, namely human apoproteins. Based upon the applicants'identification of a new lipoprotein fraction isolated from blood sampleswithdrawn from subjects post-SUV infusion, preferred apoproteins areclass A and C apoproteins, and particularly apo A-1 and apo Cs.

Apoproteins, a component of naturally-occurring lipoproteins, may berecovered from samples of the corresponding lipoprotein containing suchapoprotein(s), typically derived from either human or rat. In anexemplary procedure, the desired lipoprotein fraction is first isolatedfrom plasma by ultracentrifugation (Hatch, 1968; Chung, 1986) using anappropriate density gradient, followed by purification, typicallydialysis.

Typical sources for the A and C apoproteins are HDL and VLDL, althoughany lipoprotein containing A and C apoproteins is suitable. Generally,high density lipoproteins are isolated by ultracentrifugation atdensities from 1.063-1.21 g/ml (Havel, 1955), followed by washing byreflotation at a density of 1.21 g/ml.

Isolation of HDL₃, one potential source of apo A-1, has also beendescribed (Brissette, 1986). Briefly, human HDL₃ is prepared bysequential ultracentrifugations between densities 1.125 and 1.21 g/mland isolated at density 1.21 g/ml (4.6×10⁶ g-h). The HDL₃ is then washedunder similar conditions, followed by dialysis against a saline solutioncontaining EDTA and sodium azide.

VLDL, from either rat serum or human plasma, is similarly isolated usingultracentrifugation with an appropriate density gradient. In a typicalprocedure, VLDL is isolated from human plasma by ultracentrifugation at8.5×10⁴ g for about 0.5 h to remove chylomicrons, followed by two roundsof ultracentrifugation at 1.8×10⁶ g-h to isolate and wash the VLDL(Brissette, 1986).

Isolation of apoproteins from the corresponding lipoprotein fraction canbe carried out by a number of suitable techniques. Both apo A-I and A-IVcan be recovered from HDL. In one such method, apo A-1 and A-IV areisolated from HDL by gel filtration and ion-exchange chromatography(Edelstein, 1972; Rifici, 1985). Apo A-1 may also be extracted from asolution of human HDL₃ by precipitation with acetone, followed bycentrifugation (Brissette, 1986). In a similar fashion, apo E and apo Cscan be isolated from VLDL using selective extraction procedures(Holmquist, 1977).

Generally, isolated apoprotein fractions are identified by eitherimmunodiffusion analysis with antibodies against the targetapoprotein(s) (Bisgaier, 1987) or by polyacrylamide gel electrophoresis.A suitable gel system for carrying out electrophoretic analysis is 4-18%polyacrylamide gradient gel in the presence of sodium dodecyl sulfateand β-mercaptoethanol.

A typical polyacrylamide gel electrophoretic mobility pattern for amixture containing apo E, apo A-I, apo A-II, apo Cs and apo Bs reveals aprogression, taken from the origin, as follows: apo Cs and A-II, apoA-I, apo E, apo B₁ and apo B_(h), as provided in Brissette (Brissette,1986).

In some instances, the isolated apolipoproteins may optionally contain aradiolabel such as iodine-125. Radiolabelled apoproteins may be usefulin initial experiments for forming the lipoprotein particles of thepresent invention by providing a means for estimating the level ofapoprotein-SUV association.

Alternatively, apoproteins for use in the present invention may beformed recombinantly. Expression of apoprotein genes is known in theart. Human apo C-II can be prepared by cloning a full length human apoC-II cDNA insert into the pSP19 expression vector, followed bytranscription and translation in vitro (Holtfreter, 1988). Mature apoCII can be expressed in E. coli transformed with the pKK233-2 apo CIIclone, or alternatively, can be formed on a preparative scale byintegrating apo CII cDNA into the pUR291 vector (Holtfreter, 1988).

Expression of human apo A-I and apo A-II genes in Xenopus laevis ooctyeshas also been described (Haase, 1988). In following the method of Haase,apo A-I and apo A-II genes can be isolated as clones and transcribed andtranslated in Xenopus laevis oocytes. Simultaneous injection of the apoA-I and apo A-II genes leads to secretion of both apoproteins, which maythen be separated by density gradient centrifugation as has beendescribed. Apo A-1 can also be produced in Chinese hamster ovary (CHO)cells by transfection of the CHO cells with an expression plasmid whichplaces the human apo A-I gene under the direction of the humanmetallothionein II gene promoter (Mallory, 1987).

E. Forming the Lipoprotein Composition

IN VITRO METHOD

The therapeutic lipoprotein composition provided by the presentinvention contains lipoprotein particles of the invention composed ofsmall unilamellar vesicles of phosphatidylcholine phospholipids havingassociated A and C apoproteins. The lipoprotein particles of the presentinvention may be prepared by both in-vitro and in-vivo methods, as willherein be described.

In accordance with one method of the invention, the lipoproteinparticles are formed by mixing phosphatidylcholine SUVs with apoproteinsunder conditions suitable for forming apoprotein-associated SUVs. In onesuch exemplary approach, PC liposomes prepared as described above areincubated with apoprotein. Typically, apoprotein is incubated with theSUVs for a period of at least 1 hour and preferably for 4-8 hours, attemperatures between about 25° C.-37° C. To determine the appropriateconcentration of apoprotein, the incubation is typically performed atseveral different concentrations of protein and the degree ofassociation is determined, based upon the amount of free apoproteinremaining. For each apoprotein contained in the mixture, theprotein/phospholipid ratio will typically range from about 0.1-1 (on aμg-to μg basis), with concentrations of SUVs and protein contained inthe mixture between about 10-80 μg/ml.

Apoproteins for use in forming the lipoprotein particles of the presentinvention include apo As (primarily apo A-1, apo A-II, apo A-IV) and apoCs (primarily apo C-I, apo C-II, apo C-III). In one embodiment, thelipoprotein particles contain apo A-I and apo Cs. The apoproteins may bederived from extracted lipoprotein sources or may be recombinantapoproteins, as described above.

Returning to preparation of the lipoprotein particles, excess ornon-associated apoprotein is removed from the mixture, typically bywashing followed by centrifugation. Following verification of purity,the resulting suspension of lipoprotein particles may be stored as asuspension, or alternatively, the lipoprotein particles may be isolated.

Isolation of the newly formed lipoprotein particles is carried out usinga Ficoll gradient flotation protocol. In one such exemplary isolationmethod, the lipoprotein particles are mixed with a Ficoll solution inphosphate-buffered saline and placed in a centrifuge tube. The solutionis then overlayed with Ficoll, followed by a layer of phosphate-bufferedsaline. The resulting mixture is then centrifuged. Lipoprotein particlesfloated to the saline and Ficoll layers are collected and the procedureis repeated. The recovered liposomes may be analyzed by SDS-PAGE,typically carried out on acrylamide gel under reducing conditions. Basedupon analysis of the resulting electrophoretic patterns, the lipoproteinparticles of the present invention are identified by a distinct bandhaving an electrophoretic mobility and density distinct from those ofthe reaction components and from naturally-occurring lipoproteins. Thelipoprotein particles of the present invention are characterized bydensities between those of VLDL and LDL, namely between 1.0006 and 1.019g/ml. The ratio of protein/phospholipid (m/m) in the isolatedlipoprotein fraction will generally be between about 0.05-0.5 for eachof the apoproteins present.

Protein levels in the recovered lipoprotein particles can be verifiedusing the method of Lowry (Lowry, 1951). Phospholipid concentrations canbe determined as described in Example 5 or by digestion with perchloricacid to promote phosphate cleavage (Fiske, 1926).

In an alternate embodiment, the lipoprotein particles are formed byincorporating apoprotein directly during SUV preparation. In thisapproach, SUVs are prepared and sized as described in Examples 1 and 2,with the exception that apoprotein is included with the vesicle-forminglipids used to form the initial lipid film. Protein is added at aprotein/phospholipid (mass/mass) ratio typically between about 0.1-1.

Alternately, the cholate-lipid dispersion method can be used to complexpurified A and C apoproteins to egg PC liposomes containing cholesterol(Matz, 1982).

IN VIVO METHOD

In accordance with another aspect of the present invention, thelipoprotein particles of the invention are formed by intravenouslyadministering small unilamellar vesicles to a subject, allowing theliposomes to circulate in the bloodstream, and optionally isolating froma blood sample withdrawn from the subject, the lipoprotein particles ofthe invention.

Preparation of lipoprotein particles according to this aspect of theinvention is supported in Examples 3 and 6, and will be furtherdescribed below.

In this approach, small unilamellar vesicles composed ofphosphatidylcholine, as described in Examples 1 and 2 and sections IIA-Cherein, are intravenously administered to an animal or human subject.The vesicles may be administered in a single dose, or in multiple doses.The amount of liposomes administered at each dose is between about 10and 1000 mg lipid per kg of body weight, and preferably between about50-1000 mg lipid per kg of body weight. After allowing the injectedliposomes to circulate in the bloodstream for a period of several hours,preferably at least 2 hours, e.g., 6-24 hours, a blood sample is removedfrom the subject. The lipoprotein particles of the present invention maybe isolated from serum and used as described above. The lipoproteinparticles form in the bloodstream.

The post-infusion lipoprotein particles are typically separated fromnaturally occurring lipoproteins by ultracentrifugation, the method ofwhich has been previously described herein. The lipoprotein particlesare purified by gel electrophoresis, typically using a gradient ofpolyacrylamide. The lipoprotein particles of the invention, as formed byintravenous administration of PC SUVS, are characterized by a densitybetween that of VLDL and LDL, namely between about 1.0006 and 1.019g/ml.

III. Methods of Treating Disease States

This section describes treatment methods which involve intravenousadministration of the lipoprotein particle composition described above.In all of these methods, the composition is administered intravenouslyat in a dose and dosing frequency effective to produce a desiredimprovement in the treated condition.

A preferred dosing frequency is one-two times per week. The dosingperiods, e.g., two weeks, may be interrupted by a wash-out period,typically of 1-4 weeks. The treatment, e.g., involving repeating dosingand wash-out periods, may continue over an extended period of severalmonths or more.

The amount of composition administered at each dose is preferablybetween about 1 to 50 mg vesicle lipid per kg of body weight. In apreferred embodiment, the liposome suspension is administered one timeper week, at a dose of about 5-20 mg lipid/kg body weight.

A typical dose for an 80 kg individual would be between about 0.4 to 1.6grams lipid, corresponding to between 2-8 ml of a particle suspensioncontaining 200 ms lipid/ml. Administration may be by iv (intravenous)injection, or iv drip (infusion). The particle composition may besuspended in sterile saline or in a nutritional or drug-containingbuffer or medium, such as a glucose/salt medium, to combine liposometreatment with other parenteral therapy.

A. Treatment of SUV-responsive disease states

The applicants have previously demonstrated the ability to treat anumber of disease conditions, including acute renal failure andhypertension, by intravenous infusion of SUV's of the type employed inthe preparation of the present particle composition.

In the present invention, these conditions and disease states aretreated by intravenous administration of the serum particles like thoseformed with IV administration of such SUV's, as demonstrated above. Theadvantage of the present invention over IV administration of precursorSUV's is threefold:

First, the apoprotein particles can be expected to form in vivo onlyafter a several hour period, and thus a substantial percentage ofadministered SUV's is cleared before effective apolipoprotein particleformation occurs. Thus, substantially less liposome lipid is required inthe present invention, where the particles are active immediately onadministration.

Secondly, the activity of the lipoprotein particles formed in vivo islimited by the amount of serum apoprotein available for binding to theadministered liposomes. The present composition can be produced withrelatively high concentrations of apoproteins, permitting therapeuticeffectiveness at significantly lower amounts of administered liposomallipid.

Finally, the composition of the invention does not rely on depletingexisting serum apoproteins A and C, either in free or HDL form, from thebloodstream, so that the administered liposome/apoprotein particles tendmore to augment, rather than replace, native HDL particles.

In one embodiment, the method is employed for use in treating acuterenal failure (ARF) in a subject, as evidenced by elevated levels ofserum creatinine. ARF is typically detected by determination ofglomerular filtration rate (GFR) or blood urea nitrogen or serumcreatinine levels. GFR is the rate of ultrafiltration of plasma acrossthe walls of the glomerular capillaries and measurement of total GFR ofboth kidneys provides a sensitive index of overall renal excretoryfunction. Normal renal excretory function is indicated by a GFR of about125 mL/min (180 L/day), although when renal excretory capacity isimpaired, total GFR declines.

Often, measurements of urea and creatinine concentrations are used toassess the glomerular filtration rate. Both substances are produced at arelatively constant rate by the liver and muscles; an increase in theirrespective serum concentrations occurs as GFR declines due to the factthat both compounds undergo complete glomerular filtration and are notreabsorbed by the renal tubules. Creatinine provides a more reliableindex of GFR than urea because urea can back diffuse more completelyfrom tubule lumen to peritubular blood than creatinine.

Chemical analysis of both urine and serum samples are useful indicatorsof ARF. For example, the range of urine osmolalities that can beachieved by an individual with normal-functioning kidneys (40 to 1200mosmol/kg) is much larger than the range achievable in diseased kidneys(250-350 mosmol/kg). Typically, acute renal failure is characterized byurine osmolalities of below about 400 mosmol/kg, urine sodiumconcentrations above about 40 mmol/L, a ratio of urine-to-plasmacreatinine levels below 20, and a fractional excretion of filteredsodium, defined as the ratio of urine sodium concentration/serum sodiumconcentration to urine creatinine concentration/serum creatinineconcentration multiplied by 100, of about 2 (Harrison).

In the present embodiment, a person having acute renal failure, asevidenced by a ratio of urine-to-plasma creatinine levels less thanabout 20, is treated by iv administration of the lipoprotein compositionof the invention, as described above. The therapeutic effect of thetreatment is monitored by assaying urine-to-serum creatinine levels (orother characteristic of ARF discussed above). Treatment is continued,i.e., with repeated administration 1-2 times/week, until a significantimprovement (rise) in urine-to-serum creatinine level is achieved.

In another embodiment, the method is employed for the treatment ofhypertension. Hypertension refers to elevated arterial pressure, and istypically reported as a ratio of systolic pressure (arterial pressureduring contraction of the heart muscle) to diastolic pressure (residualarterial pressure during relaxation of the heart muscle), reported inunits of mm Hg. A normal diastolic blood pressure is between about 60-85mm Hg. Diastolic pressures above 85 mm Hg are generally diagnostic ofhypertension.

In the present invention, a subject having elevated blood pressure,i.e., diastolic pressure above about 85 mm Hg, is treated by ivadministration of the lipoprotein particles of the invention.Therapeutic effectiveness is followed by monitoring blood pressure,preferably diastolic blood pressure. Treatment is continued until, e.g.,by repeated administration of the composition 1-2 times/week, until asignificant reduction, and preferably at least a 10% reduction indiastolic blood pressure is observed.

B. Treatment of Disease States Associated with Elevated Lp(a)

In another general embodiment, the invention provides a method ofreducing the serum Lp(a) concentration in a person at risk fordeveloping a disease condition associated with a chronic elevated serumLp(a) concentration. Conditions associated with elevated Lp(a)concentrations include, for example, gout, breast cancer andhyperthyroidism.

A chronically elevated Lp(a) concentration refers to a serum Lp(a)concentration that is above about 25 mg/dl, typically representing anaverage of Lp(a) values, when measured several times over the course ofa week. Serum Lp(a) concentrations can be measured by a variety ofmethods, including enzyme-linked immunoabsorbent assay (ELISA), lateximmunoassay or immunoradiometric assay. A specific kit for determiningLp(a) concentration in a blood sample, Macra™, is available from Terumodiagnostics (Elkin, Md.).

In the present invention, a subject having elevated Lp(a) level, i.e., aserum Lp(a) level above 25 mg/dl, Hg, is treated by iv administration ofthe lipoprotein particles of the invention. Therapeutic effectiveness isfollowed by monitoring serum Lp(a) level, and treatment is continueduntil, e.g., by repeated administration of the composition 1-2times/week, until a significant reduction, and preferably at least a20-40% reduction in serum Lp(a) level is observed.

In more specific embodiments, the invention includes a method oftreating gout, breast cancer or hyperthyroidism in a subject having oneof these conditions and an elevated serum Lp(a) concentration. Studieshave shown that serum Lp(a) concentrations are elevated in many subjectswith gout (Takahashi), various types of cancer, such as breast cancer(Kokoglu), and hyperthyroidism (Yamamoto). The purpose of this method isto treat the clinical disease by lowering Lp(a) levels, as one of theunderlying factors contributing to the disease.

Treatment, in accordance with the method of the invention, involvesfirst determining serum Lp(a) concentration in a person having one ofthe above clinical conditions. A patient having an elevated Lp(a) levelis then selected as a candidate for the liposome treatment method, asdescribed above, typically as an adjunct to another treatment method,such as surgery, chemotherapy, or radiation therapy in the case ofbreast cancer. Treatment is maintained until a significant reduction inLp(a) is observed and preferably throughout the treatment period for theclinical disease.

C. Treatment of Restenosis

Restenosis occurs in approximately 20-30 percent of patients followingpercutaneous transluminal coronary angioplasty. Restenosis can alsooccur in patients following surgical resectioning of vascular tissue. Inthis procedure, a region of stenosis in a vessel is removed and thevessel is sutured closed. Restenosis in each case is apparently theresult of excessive local myointimal hyperplasia, brought about byplatelet aggregation to the freshly dilated or sutured vessel surface(Harrison).

Recent studies have shown that high serum Lp(a) concentrations areassociated with an increased incidence of restenosis after balloonangioplasty (Daida, Desmarais, Tenda). In one study, patients with aserum Lp(a) level of 38 mg/dl had a significantly higher level ofrestenosis than patients with a serum level of 19.9 mg/dl (Tenda).

The present invention includes a method of reducing the extent ofrestenosis following procedures such as balloon angioplasty or surgicalresectioning of vascular tissue. Typically in the method, a personundergoing such a procedure that can lead to restenosis is given one ormore pretreatment administrations of the lipoprotein particlecomposition, particularly where existing Lp(a) levels are elevated, toachieve a reduction in such levels. Following the procedure, the patientis again monitored for Lp(a) serum concentrations, and given furtherparticle injections if necessary to maintain or achieve low Lp(a)levels, e.g., 20 mg/dl or lower. The treatment may be discontinued aftera period of several weeks or more when the risk of restenosis haspassed.

The following examples illustrate, but in no way are intended to limitthe scope of the present invention.

MATERIALS AND METHODS

Egg phosphatidylcholine (egg PC) recovered from egg yolk may be preparedaccording to known methods (Shinitsky, et al., 1974). High purity egg PCmay also be purchased from Avanti Polar Lipids (Alabaster, Ala.), LipoidKG (Ludwigshafen, Germany), or from Sigma (St. Louis, Mo.). The egg PCused to form small unilamellar vesicles as described below wasdetermined to be greater than 99% pure by thin layer chromatography(TLC) analysis.

EXAMPLE 1 Preparation of Small Unilamellar Vesicles: Sonicaiton

Egg PC dissolved in chloroform was placed in a 100 ml vessel and driedto a thin film under an inert atmosphere of nitrogen. Sterile saline wasadded to the lipid film to a final concentration of about 100 mg/ml, andthe lipid film was hydrated with swirling. The resulting multilamellarvesicle (MLV) suspension was then bath sonicated for 1 hour using a HeatSystem Sonicator, Model 375W, at a power setting of 40-50% full value.The temperature of the suspension was maintained at about 4° C. duringsonication. Large vesicles or MLVs were separated from the sonicatedsuspension by ultracentrifugation at 100,000 g for 1 hour (Barenholz,1977). The remaining suspension of SUVs, having a concentration of about100 mg/ml, was then filter sterilized.

EXAMPLE 2 Preparation of Small Unilamellar Vesicles: Extrusion

Homogeneous small unilamellar vesicles of egg PC with an averagediameter of 39±8 nm, in 0.15 M NaCl were prepared by extrusion usingserial filtration through polycarbonate filters in a GH 76-400 pressurecell (Nucleopore) (Anselem, et al., 1993). Liposomal size was determinedusing a Coulter model N4 sub-micron particle analyzer equipped with asize distribution processor analyzer (Barenholz, et al., 1993). Thefinal extrusion step was through a 0.05 micrometer pore polycarbonatefilter. Egg PC SUV's were sterilized by filtration through sterile 0.22micrometer Millipore filters.

EXAMPLE 3 Effects of Egg SUV PC Treatment on Three Male Subjects

Suspensions of small unilamellar vesicles prepared as described inExamples 1 and 2 above were administered over a 7 week period to threesubjects.

                  TABLE I    ______________________________________    Subject No.      Gender  Age    ______________________________________    (1)              male    40    (2)              male    54    (3)              male    64    ______________________________________

The following treatments were administered to each of the threesubjects:

                  TABLE II    ______________________________________    Treatment No.             Week    Treatment Regime    ______________________________________    1        1       i.v. infusion of 250 ml of 0.9% NaCl                     solution, followed by i.v. infusion of 200                     mg SUVs/kg body weight    2        2       i.v. infusion of 300 mg SUVs/kg body weight    3        3       i.v. infusion of 300 mg SUVs/kg body weight    4        4       i.v. infusion of 300 mg SUVs/kg body weight    --       5-8     i.v. administration suspended    5        9       treatment resumed with i.v. infusion of 300                     mg SUVs/kg body weight    6        10      i.v. infusion of 300 mg SUVs/kg body weight    7        11      i.v. infusion of 300 mg/kg SUV/kg body                     weight    ______________________________________

Over the course of treatment, pulse rate, blood pressure, bodytemperature and body weight were monitored and no significant changeswere observed.

None of the subjects reported hypersensitivity reaction or other adverseeffects relating to SUV treatment.

Subject No. (1) reported an improvement in skin texture, noting that hisskin appeared to be more "silky". Subject No. (3) reported animprovement in his ability to perform strenuous physical activity and inthe condition of his gums.

Laboratory test results for each of the subjects revealed noabnormalities in liver function, renal function, glucose, electrolytes,CPK and aldolase levels. Complete blood count, coagulation tests andblood hormone levels (e.g., thyroid stimulating hormone (TSH), cortisoland testosterone) were within normal ranges and did not changesignificantly over the course of treatment. EKG traces for each of thesubjects were normal over the course of treatment and abdominalultrasound performed before and after completion of the study werenormal and revealed no indications of fatty liver.

EXAMPLE 4 Osmotic Fragility of Red Blood Cells

The osmotic fragility of red blood cells from each of subjects (1)-(3)was determined over the period of SUV treatment.

Hypotonic solutions of sodium chloride at pH 7.4 were used to determinethe concentration at which cell lysis occurred for both fresh red bloodcells and for red blood cells which were incubated for 24 hours at 37°C. The concentration of sodium chloride at which lysis occurred in 50%of the cells (e.g., the median corpuscular fragility or MCF) was used asan indication of cell osmotic fragility.

Red blood cell samples (both pre and post infusion) exhibited MCF valuesat 20° C. and pH 7.4 within a concentration range of 4.0-4.5 g NaCl/mlfor fresh red blood cells and between 4.65-5.9 g NaCl/ml for red bloodcells incubated at pH 7.4 at a temperature of 37° C. for 24 hours.

The MCF values for all samples remained in the normal range over thecourse of the study.

EXAMPLE 5 Rate of Appearance and Clearance of SUVS in the Circulation

Serum phospholipid levels were determined in order to follow the rate ofappearance and clearance of intravenously administered liposomes in thecirculation.

Serum isolated from blood samples taken from each of the subjects atvarious times after SUV infusion were extracted by standard methods.Phosphatidylcholine concentrations in blood following liposome injectionwere determined by measuring the increase in inorganic phosphate (P_(i))in serum. PC concentrations determined based upon determination ofinorganic phosphate were confirmed by an enzymatic assay specific forcholine phospholipids.

PC levels in serum reached maximal levels at the end of the infusionperiod, decreased considerably over the next 24 hours following infusionand gradually increased to preinfusion values at day 8. Based upon thisfinding, the treatment regime described in Table 2 (e.g., infusion every7 days) was followed.

EXAMPLE 6 Isolation and Characterization of Lipoprotein Particles

Liposomes were recovered from blood samples withdrawn from the patientsat various times after infusion and isolated by centrifugation at adensity of 1.006 g/ml or 1.019 g/ml. The recovered post-infusionliposomes were characterized as a distinct lipoprotein fraction justabove LDL.

The post-infusion liposomes in the isolated lipoprotein fraction werethen examined by electron microscopy and observed to form bilayers.Electron micrographs of negatively stained LDL and VLDL fractionsrevealed that both the LDL and VLDL fractions also contained liposomes.

The composition of the recovered lipoprotein fraction was determined bySDS-PAGE on acrylamide gel. Based upon electrophoretic mobility, theisolated lipoprotein fraction was determined to contain Apo A-1, Apo E,and Apo Cs. Apo B was not detected. The recovered lipoprotein fractionwas also found to contain free cholesterol. The composition andcharacteristics of the recovered lipoprotein fraction are summarized inTable III below.

                  TABLE III    ______________________________________    New Lipoprotein Fraction    Density (g/ml)     between 1.006-1.019    ______________________________________    Electron microscopy                       bilayers                       size estimate?    Apoproteins:       Apo A-1                       Apo Cs                       Apo E    Lipids             phospholipid                       free cholesterol    ______________________________________

EXAMPLE 7 Post SUV Treatment: Plasma Analysis

Plasma isolated from blood samples taken from each of the subjects atvarious times after SUV infusion was analyzed for free cholesterolcontent, levels of LDL-C, HDL-C, Apo A-1, triglycerides, and Lp(a).

Twenty four hours after infusion with SUVs, red blood cell freecholesterol levels were found to be reduced by 27%, 10.5% and 18% insubjects No. (1), (2), and (3), respectively, and returned to baselinevalues 24 hours later. The change in red blood cell cholesterol contentfollowing SUV infusion did not affect red blood cell osmotic fragility.No signs of homolysis were observed, as indicated by constant levels ofserum haptoglobin and free hemoglobin observed both before and after SUVtreatment.

Post SUV infusion serum samples were centrifuged at a density of 1.019g/ml to isolate the LDL-C fraction. No significant change in thepost-infusion LDL-C fraction was observed.

Plasma analysis also revealed that during the course of treatment,profiles of HDL-C and apo A-1 levels tended to rise. Triglyceride levelsremained substantially unchanged. Lp(a) levels were found to besubstantially reduced. Levels of free cholesterol in plasma were foundto increase after SUV infusion, and returned to baseline levels as theliposomes were cleared through the circulation.

                  TABLE IV    ______________________________________    Post SUV Treatment: Plasma Analysis Summary    Free    choles-                          Trigly-    terol   LDL-C    HDL-C    Apo A-1                                     cerides                                            Lp(a)    ______________________________________    increased            no change                     increased                              increased                                     no change                                            reduced    ______________________________________

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

It is claimed:
 1. A method of reducing serum Lp(a) concentration in asubject at risk for developing a disease condition associated withchronic, elevated serum Lp(a) concentrations, comprising intravenouslyadministering to the subject a suspension of small unilammelar liposomescomposed primarily of phosphatidylcholine phospholipids having phasetransition temperatures in the range of about -10 and 37° C. over aperiod of time and in an amount effective to result in a significantreduction in serum Lp(a) concentration.
 2. The method of claim 1,wherein the liposomes have a size in the range of about 0.02 and 0.12microns.
 3. The method of claim 1, wherein the phosphatidylcholine isegg phosphatidylcholine.
 4. The method of claim 1, wherein the phasetransition temperatures are less than about 5° C.
 5. The method of claim1, wherein the liposomes are administered in a dose of about 500 mglipid/kg body weight.
 6. A method of treating gout, breast cancer orhyperthyroidism in a subject having an elevated serum Lp(a)concentration, comprising intravenously administering to the subject asuspension of small unilammelar liposomes composed primarily ofphosphatidylcholine phospholipids having phase transition temperaturesin the range between about -10 and 37° C. over a period of time and inan amount effective to result in a significant reduction in serum Lp(a)concentration.